The present invention relates to the modification of biological molecules for the purpose of improving their efficacy as therapeutic treatments.
Traditionally, therapeutic proteins have several inherent shortcomings. Proteins often have short half-lives, wide tissue distribution, the potential for immunogenicity, and sometimes need to be dosed frequently. When frequent dosing is required, it can result in increased cost, toxicity and complicated dosing regimens. In an effort to overcome these shortcomings, researchers have looked at improving the delivery systems of proteins. Among the potential solutions lies PEGylation, the attachment of a flexible strand or strands of polyethylene glycol (PEG) to a protein.
When attached to a drug or protein, PEG polymer chains can sustain bioavailability by protecting the drug molecules from immune responses and other clearance mechanisms. PEGylation has been shown to favourably alter pharmacokinetics by prolonging circulation time and decreasing clearance rates, to delay absorption, decrease systemic toxicity and to display increased clinical efficacy (partly by exhibiting reduced proteolysis).
Clinical developments are reported for PEG conjugates of proteins, peptides, aptamers, natural products and small molecules. For instance, PEG-asparaginase has been used in Oncaspar™ by Enzon to treat acute lymphoblastic leukemia and PEG-α-interferon 2b has been used by Schering Plough to treat Hepatitis C.
WO 2005/007197 describes a series of novel reagents which can be used, inter alia, to conjugate thiol groups of two cysteine residues in a protein to give novel thioether conjugates. The reagents comprise polymers which may be, for example, a polyalkylene glycol, a polyacrylate or a HPMA polymer. It is disclosed that interferons may be conjugated, and their biological activity retained compared with non-conjugated interferons.
Overall, PEG attachment to interferon-alpha leads to a longer half-life of the interferon. This occurs due to decreased clearance by the kidney and reduced proteolysis (slower breakdown of protein). In addition, PEG attachment leads to lowered antigenicity of interferon. PEG attachment also leads to increased chemical and thermal (heat) stability of the base substance interferon.
PEGylation, however, has its disadvantages. PEGylation of proteins is known for its suboptimal yields; losses of 20-40% of protein and PEG-agent are not uncommon. Many of the common linking technologies are non-specific and the protein can be PEGylated at multiple sites in a random fashion, producing a mixture of products with variable activities. It is important, for optimised efficacy to ensure that the number of conjugated polymer molecules per protein is the same and that each polymer is attached to the same residue in each protein molecule.
Some have tried to avoid such problems by attaching PEG to the N-terminal amino group of proteins and peptides. This is called N-terminal PEGylation. N-terminal PEGylation may offer advantages in purification of the conjugates. It is also believed that the N-terminal PEGylation may better preserve bioactivity as compared to a random PEGylation of amino group of lysine residues.
Thiol specific polymer conjugating reagents for proteins have been developed. These are generally more hydrolytically stable than their amino-specific counterparts and thus can be used at lower stoichiometric excess. Conjugating functional moieties that are broadly selective for thiol groups include iodoacetamide, maleiimide (WO 92/16221), vinylsulfone (WO 95/13312 and WO 95/34326), vinyl pyridines (WO 88/05433), and acrylate and methacrylate esters (WO 99/01469). These thiol selective conjugating moieties yield a single thioether conjugating bond between the polymer.
Conjugation to a protein via a thiol residue is also advantageous since proteins typically contain few thiol groups, hence conjugation can be specifically directed to a certain residue or residues on each protein.